Nitrones behave as spin trapping agents when a diamagnetic nitrone compound (the “spin trap”) reacts with a transient free radical species (having the “spin”) to provide a relatively more stable radical species (referred to as the “spin adduct”). The spin adduct may be detectable by electron paramagnetic resonance (EPR) spectroscopy if the spin adduct has a reasonable lifetime. Thus, information about the spin can be gleaned from a study of the structure and spectroscopic characteristics of the spin adduct. For example, the toxicity of synthetic beta-amyloid peptide preparations toward glutamine synthetase could be correlated with the characteristics of the EPR signal generated by the spin adduct formed from each batch of synthetic beta-amyloid peptide and the spin trap PBN. See, Hensley, K. et al., in NeuroReport (1995) 6:489-492. Beta-amyloid peptides are neurotoxic substances that are postulated to be involved in the etiology of Alzheimer's disease.
Low molecular weight nitroxides are non-immunogenic. Moreover, they are typically cell permeable and can exist as a non-toxic, stable free radical capable of partitioning among various cellular compartments. Being paramagnetic, nitroxides are detectable by electron paramagnetic resonance (EPR) spectrometry and may serve as contrast agents in magnetic resonance imaging (MRI). See, Brasch, R. C, in Radiology (1983) 147:781; Keana, J. F. and Van, N. F., in Physiol. Chem. Phys. Med. NMR (1984) 16:477. Nitroxides have also been used as biophysical markers to probe cellular metabolism, oxygen level, intracellular pH, protein/lipid mobility and membrane structure. Hence, nitroxides find use in a number of diagnostic methods to determine the physiological/medical condition of a subject or the biophysical characteristics of a given sample, including samples obtained from a biological fluid.
Free radicals and oxidative damage have been implicated in brain aging and several neurodegenerative diseases. See, Socci, D. J. et al., in Brain Research (1995) 693(1-2):88-91. Chronic treatment of aged rats with certain compounds, including the spin, trapping agent alpha-phenyl N-tert-butylnitrone (PBN) and the antioxidant alpha-tocopherol (vitamin E), was found to benefit (i.e., improve) age-related changes in cognitive performance.
In vitro and in vivo evidence is mounting that the administration of antioxidants can strongly reduce the rate of progression of lesion formation associated with the process of atherosclerosis. Based on several experimental models, including low density lipoprotein (LDL)-receptor-deficient rabbits, cholesterol-fed rabbits and cholesterol-fed non-human primates, several antioxidants have manifested a 50-80% reduction in the rate of progression of lesions. The effectiveness of probucol, butylated hydroxytoluene (BHI), N,N′-diphenylphenylenediamine and vitamin E are attributed to their respective antioxidant potentials and to the proposition that oxidative modification of LDL contributes to the progression of atherosclerosis. See, Steinberg, D., in Lancet (1995) 346(8966):36-38. The one-electron oxidative potential (vs. NHE) of vitamin E in an aqueous solution at pH 7 and 20° C. is 0.48 V. The oxidative potentials of PBN, POBN and DMPO range from about 1.5-2.0 V.
Further, Downs, T. R. et al, in Int'l J. Immunopharmacol. (1995) 17(7):571-580, have shown that a cyclic nitrone antioxidant, MDL 101,002, reduces organ dysfunction, and cytokine secretion induced by lipopolysaccharide (LPS) administered to rats. These authors also tested the ability of MDL 101,002 to prevent LPS-induced pulmonary edema, leukopenia and thrombocytopenia. They found that MDL 101,002 prevented pulmonary edema, partially reduced thrombocytopenia but failed to prevent leukopenia. These workers found that their results were consistent with the role that oxygen free radicals played in the development of endotoxin-induced organ dysfunction and shock. They further suggest that free radical scavengers could reduce the mortality consequent to sepsis by organ dysfunction, at least in part, through a reduction in free radical-stimulated cytokine secretion.
Allergic reactions generate reactive oxygen species, including superoxide anions, which usher the influx of inflammatory cells to the site of allergen challenge and contribute to allergic inflammation. The inflammation may, in turn, lead to cell or tissue injury. For allergic reactions in the lung, these processes are also accompanied by increased vascular permeability and changes in airway mechanics. See, Sanders, S. P. et al. in Am. J. Respir. Crit. Care Med. (1995) 151:1725-1733. Thus, the administration of spin trapping agents to the site of challenge may reduce the inflammatory response and help reduce tissue or cell damage.
Separately, oxygen-derived free radicals are suspected in playing a role in cytotoxicity during episodes of allograft rejection/destruction following infiltration of the graft by mononuclear cells. The administration of radical scavengers may thus inhibit or reduce the incidence of allograft rejection. See, Roza, A. M. et al., in Transplantation Proceedings (1994) 26(2):544-545.
New reagents that could visually signal the formation of oxidative species would be extremely useful not only in skin tests or in cell culture but also in determining, for example, the compatibility of a patient's white blood cells with a particular kidney dialysis membrane. In vitro calorimetric assays would be of great utility.
PBN has been shown to offer protection in the cardiovascular disease area, in particular, by trapping free radicals generated during ischemia-reperfision-mediated injury to the heart. See, e.g., Bolli, R. et al. J. Clin. Invest. (1988) 82:476. The benefits of trapping free radicals generated in similar types of injury to the brain of experimental animals has also been demonstrated. See, e.g., Oliver, C. N. et al. Proc. Nat'l. Acad. Sci. USA (1990) 87:5144; Carney, J. M. et al. Ibid. (1991) 88:3636; Floyd, R. A. Science (1991) 254:1597. Oxidative damage to protein and DNA is mediated by oxygen free radical intermediates, leading to strand breaks and base modifications. Enzymes, such as glutamine synthetase, can also be inactivated by oxidative processes. Such damage can be observed, for example, in animals subjected to brain ischemia/reperfusion injury. See Floyd, R. A. and Carney, J. M. Ann. Neurol. (1992) 32:S22-S27.
Evidence is also available that PBN inhibits oxidative modification of cholesterol and triglycerides of Low Density Lipoproteins (LDL). Oxidative modification of LDL, along with lipid peroxidation and free-radical mediated reactions, is a process that is implicated in the initiation of atherosclerosis. See, e.g., Steinberg, D. et al., N. Engl. J. Med. (1989) 320:915; Esterbauer, H. et al. Ann. N.Y. Acad. Sci. (1989) 570:254.
Free radicals and oxidative damage have been proposed as the underlying reasons for aging, chronic and degenerative diseases of aging, and acute clinical conditions. Daily administration by intraperitoneal injection of PBN to an aged animal model showed that PBN offered a remarkable extension of the lifespan in both male and female populations. See, Packer, L. et al., in Biochem. Biophys. Res. Commun. (1995) 211(3):847-849. These authors conclude that PBN could have prophylatic value against the onset of, at least, pathological senescence.
Bruce N. Ames and co-workers, in an article published in the Proc. Nat'l. Acad. Sci. USA (1995) 92:4337-4341, found support for the hypothesis that oxidative DNA damage contributes to replicative cessation in human diploid fibroblast cells. These workers found that senescent cells, those cells that have ceased growth in culture after a finite number of population doublings, excise from DNA four times more 8-oxoguanine per day than do early-passage young cells. Also, levels of 8-oxo-2′-deoxyguanosine in DNA of senescent cells are about a third higher than those found in DNA of young cells. Most interestingly, they found that PBN, perhaps acting as either an antioxidant or as a spin trapping agent, effectively delayed the onset of senescence and rejuvenated near senescent cells.
International Application No. PCT/US96/18570, filed Nov. 15, 1996, discloses certain chromotropic nitrone spin trapping agents, methods for making these agents, and methods for their use. These compounds are effective in trapping free radicals, and thus have utility as antioxidants in biological systems. A series of U.S. Patents has issued based on this International Application, including U.S. Pat. Nos. 6,083,988; 6,197,825; and, 6,291,702.
Additional research with these and similar compounds was published on 3 Apr. 2002 in the Journal of the American Chemical Society: Becker et al., “Stilbazulenyl Nitrone (STAZN): A Nitronyl-Substituted Hydrocarbon with the Potency of Classical Phenolic Chain-Breaking Antioxidants”, J. Am. Chem. Soc. 2002, 124:4678-84.
While the foregoing patents and related research presents, demonstrates and covers the structure and utility of a group of azulenyl nitrones, there exists a continuing need to discover additional new and effective substances exhibiting free radical/spin trapping and/or antioxidant activity which are potentially useful for a wide range of analytical, preservative, diagnostic, prophylactic and therapeutic applications. Accordingly, it is toward the satisfaction of such continuing need that the present invention is directed.